PROJECT SUMMARY Mechanical cues critically affect cell behaviors that are central to embryonic development, organ formation and the maintenance of tissue architecture and homeostasis. Both mechanical forces and the material properties of the cellular microenvironment (e.g., stiffness) are known to direct stem cell differentiation as well as alter the progression of malignant phenotypes during tumor progression. While it is generally acknowledged that mechanical forces and the material properties of the cellular microenvironment play a key role in the control of cell behaviors in vitro, the lack of technologies to perform quantitative measurements of mechanics in 3D cellular microenvironments has considerably hindered our ability to understand the role of mechanics in more physiologically relevant environments. PI Campas and coworkers have recently reported a novel methodology that enables direct in vivo and in situ mechanical measurements within 3D cellular microenvironments, including living tissues, for the first time. While groundbreaking, these methods, which make use of fluorescently-labeled, magnetically-responsive microdroplets of perfluorinated oil as mechanical sensors and actuators, remain strongly limited in scope because of current chemistry used to prepare the microdroplets. The current finicky chemical composition of the microdroplets strongly limits the scope of the technique, hampers the reproducibility of the measurements and precludes the dissemination of these methods to the wide biological and biomedical communities. In this multi-PI technology development grant, Campas, Sletten, and Zink team up to solve the existing problems with the microdroplet technology by developing new robust chemistries, including fluorinated surfactants, fluorophores, and magnetic nanoparticles, to enable accurate mechanical measurements with microdroplets in a wide range of biological systems, from living tissues and organs to organoids, tumors and 3D cell culture. In Aim 1, we will develop surfactants and fluorophores to properly control the droplet?s interfacial tension, the cell droplet interactions and their visualization in 3D multicellular systems. In Aim 2, we will develop robust perfluorocarbon-based ferrofluids with controlled interfacial tension, cell-droplet interactions and with strong magnetic properties enabling the application of larger forces. In Aim 3, we will test the functionality of the newly synthesized compounds and assess their performance in mechanical measurements in well-established 3D multicellular systems, both in vitro and in vivo. Upon completion of these aims, we will achieve an optimized microdroplet technology that is ready for commercialization and can be used in a wide range of systems, including living tissues, organoids, embryoid bodies, tumors and 3D cell culture, thereby making accessible these new tools for the study of mechanical cues (mechanobiology) in vivo to the entire biological and biomedical communities and potentially transforming our understanding of biological systems.